Modification of 5′-UTR sequences of pPIC9 increases expression of antimicrobial peptide LL-37 / 第二军医大学学报

ABSTRACT

Objective:To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris.Methods:The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was trans- formed into E.coli DH5?to construct a modified eukaryotic vector pPIC9-EDIT.After PCR and sequencing,pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast,the product was then transformed into E.coli DH5?to con- struct the recombinant expression vector pPIC9-EDIT-LL-37,the latter was transformed into P.pastoris GS115 by spheroplas- ting and the insert was confirmed by PCR.The bacteriolytic activity to E.coli.DH5?was analyzed to screen the highest ex- pressing strain and to determine the best inducing time and concentration of methanol.The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting.The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-ED- IT-LL-37 were compared,and the changes of LL-37 protein expression were determined before and after modification.Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed.Expression of LL-37 gene was confirmed by PCR in P.pastoris after pPIC9-EDIT-LL-37 transformation.The highest expressing strain was identified;the best inducing time was 72 h and the best concentration of methanol was 0.5%.Tricine-SDS-PAGE and Western blotting analysis showed that the ex- pression product was LL-37.The expression level of LL-37 protein increased by 35 times after modification.Conclusion:Modifi- cation of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P.pastoris;it is worth to be used in the research of other heterogenous protein.